This invention relates to compositions and methods useful for the prevention, diagnosis and treatment of Lyme disease. More particularly, this invention relates to novel B. burgdorferi polypeptides which are able to elicit in a treated animal, the formation of an immune response which is effective to prevent or lessen the severity, for some period of time, of B. burgdorferi infection. This invention also relates to multicomponent vaccines comprising one or more of the novel B. burgdorferi polypeptides. Also within the scope of this invention are antibodies directed against the novel B. burgdorferi polypeptides and diagnostic kits comprising the antibodies or the polypeptides.
Lyme borreliosis is the most common vector-borne infection in the United States [S. W. Barthold, et al., xe2x80x9cAn Animal Model For Lyme Arthritisxe2x80x9d, Ann. N.Y. Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.
The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.
Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al., xe2x80x9cIsolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migransxe2x80x9d, J. Am. Acad. Dermatol., 3, pp. 444-49 (1985)]. Flu-like or meningitis-like symptoms are common at this time. Stage two occurs within days or weeks, and involves spread of the spirochete through the patient""s blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., xe2x80x9cBorrelia burgdorferi-Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitisxe2x80x9d, Ann Neurol., 24, pp. 509-16 (1988)].
B. burgdorferi is much easier to culture from the tick than from humans, therefore at present, Lyme disease is diagnosed primarily by serology. The enzyme-linked immunosorbent assay (ELISA) is one method of detection, using sonicated whole spirochetes as the antigen [J. E. Craft et al., xe2x80x9cThe Antibody Response In Lyme Disease: Evaluation Of Diagnostic Testsxe2x80x9d, J. Infect. Dis., 149, pp. 789-95 (1984)]. However, false negative and, more commonly, false positive results are associated with currently available tests.
At present, all stages of Lyme disease are treated with antibiotics. Treatment of early disease is usually effective, however the cardiac, arthritic, and nervous system disorders associated with the later stages often do not respond to therapy [A. C. Steere, xe2x80x9cLyme Diseasexe2x80x9d, New Eng. J. Med., 321, pp. 586-96 (1939)].
Like Treponema pallidum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A. G. Barbour and S. F. Hayes, xe2x80x9cBiology Of Borrelia Speciesxe2x80x9d, Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia burgdorferi have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane.
The B. burgdorferiouter surface proteins identified to date are believed to be lipoproteins, as demonstrated by labelling with [3H]palmitate [M. E. Brandt et al., xe2x80x9cImmunogenic Integral membrane Proteins of Borrelia burgdorferi Are Lipoproteinsxe2x80x9d, Infect. Immun., 58, pp. 983-91 (1990)]. The two major outer surface proteins are the 31 kd outer-surface protein A (OspA) and the 34 kd outer surface protein B (OspB). Both proteins have been shown to vary from different isolates or from different passages of the same isolate as determined by their molecular weights and reactivity with monoclonal antibodies OspC is a 22 kDa membrane lipoprotein previously identified as pC. [R. Fuchs et al., xe2x80x9cMolecular Analysis and Expression of a Borrelia burgdorferi Gene Encoding a 22 kDa Protein (pC) in Escherichia colixe2x80x9d, Mol. Microbiol., 6, pp. 503-09 (1992)]. OspD is said to be preferentially expressed by low-passage, virulent strains of B. burgdorferi B31 [S. J. Norris et al., xe2x80x9cLow-Passage-Associated Proteins of Borrelia burgdorferi B31: Characterization and Molecular Cloning of OspD, A Surfaced-Exposed, Plasmid-Encoded Lipoproteinxe2x80x9d, Infect. Immun., 60, pp. 4662-4672 (1992)].
Non-Osp B. burgdorferi proteins identified to date include the 41 kD flagellin protein, which is known to contain regions of homology with other bacterial flagellins [G. S. Gassman et al., xe2x80x9cAnalysis of the Borrelia burgdorferi GeHo fla Gene and Antigenic Characterization of Its Gene Product.xe2x80x9d, J. Bacteriol., 173, pp. 1452-59 (1991)] and a 93kDa protein said to be localized to the periplasmic space [D. J. Volkman et al., xe2x80x9cCharacterization of an Immunoreactive 93 kDa Core Protein of Borrelia burgdorferi With a Human IgG Monoclonal Antibodyxe2x80x9d, J. Immun., 146, pp. 3177-82 (1991)].
Recently, immunization of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with B. burgdorferi [E. Fikrig et al., xe2x80x9cLong-Term Protection of Mice from Lyme Disease by Vaccination:with OspAxe2x80x9d, Infec. Immun., 60, pp. 773-77 (1992)]. However, protection by the OspA immunogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among different B. burgdorferi isolates. For example, immunization with OspA from B. burgdorferi strain N40 confers protection against subsequent infection with strains N40, B31 and CD16, but not against strain 25015 [E. Fikrig et al., xe2x80x9cBorrelia burgdorferi Strain 25015: Characterization of Outer Surface Protein A and Vaccination Against Infectionxe2x80x9d, J. Immun., 148, pp. 2256-60 (1992)].
Immunization with OspB has also been shown to confer protection against Lyme disease but not to the same extent as that conferred by OspA [E. Fikrig et al., xe2x80x9cRoles of OspA, OspB, and Flagellin in Protective Immunity to Lyme Borreliosis in Laboratory Micexe2x80x9d, Infec. Immun., 60, pp. 657-61 (1992)]. Moreover, some B. burgdorferi are apparently able to escape destruction in OspB-immunized mice via a mutation in the OspB gene which results in expression of a truncated OspB protein [E. Fikrig et al., xe2x80x9cEvasion of Protective Immunity by Borrelia burgdorferi by Truncation of Outer Surface Protein Bxe2x80x9d, Proc. Natl. Acad. Sci., 90, pp. 4092-96 (1993)]. OspC has also been shown to have protective effects in a gerbil model of B. burgdorferi infection. However, the protection afforded by immunization with this protein appears to be only partial [V. Preac-Mursic et al., xe2x80x9cActive Immunization with pC Protein of Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infectionxe2x80x9d, Infection, 20, pp. 342-48 (1992)].
As prevention of tick infestation is imperfect, and Lyme disease may be missed or misdiagnosed when it does appear, there exists a continuing urgent need for the determination of additional antigens of B. burgdorferi and related proteins which are able to elicit a protective immune response and which may be useful in a broad-spectrum vaccine. In addition, identification of additional B. burgdorferi antigens may enable the development of more reliable diagnostic reagents which are useful in various stages of Lyme borreliosis.
The present invention provides novel B. burgdorferi polypeptides which are substantially free of a B. burgdorferi spirochete or fragments thereof and which are thus useful in compositions and methods for the diagnosis, treatment and prevention of B. burgdorferi infection and Lyme disease. In one preferred embodiment, this invention provides OspE polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides.
In another preferred embodiment, this invention provides OspF polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides.
In another preferred embodiment, this invention provides S1 polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides.
In another preferred embodiment, this invention provides T5 polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides.
The preferred compositions and methods of each of the aforementioned embodiments are characterized by novel B. burgdorferi polypeptides which elicit in treated animals, the formation of an immune response which is effective to prevent or lessen the severity, for some period of time, of B. burgdorferi infection.
In another preferred embodiment, this invention provides a multicomponent vaccine comprising one or more novel B. burgdorferi polypeptides of this invention in addition to one or more other immunogenic B. burgdorferi polypeptides. Such a vaccine is effective to confer broad protection against B. burgdorferi infection.
In yet another embodiment, this invention provides antibodies directed against the novel B. burgdorferi polypeptides of this invention, and pharmaceutically effective compositions and methods comprising those antibodies.
In another embodiment, this invention provides diagnostic means and methods characterized by one or more of the novel B. burgdorferi polypeptides, or antibodies directed against those polypeptides. These means and methods are useful for the detection of Lyme disease and B. burgdorferi infection. They are also useful in following the course of treatment against such infection. In patients previously inoculated with the vaccines of this invention, the detection means and methods disclosed herein are also useful for determining if booster inoculations are appropriate.
In yet another embodiment, this invention provides methods for identification and isolation of additional B. burgdorferi polypeptides, as well as compositions and methods comprising such polypeptides.
Finally, this invention provides DNA sequences that code for the novel B. burgdorferi polypeptides of this invention, recombinant DNA molecules that are characterized by those DNA sequences, unicellular hosts transformed with those DNA sequences and molecules, and methods of using those sequences, molecules and hosts to produce the novel B. burgdorferi polypeptides and multicomponent vaccines of this invention. DNA sequences of this invention are also advantageously used in methods and means for the diagnosis of Lyme disease and B. burgdorferi infection.